Numerous cell surface receptors transduce signals through heterotrimeric GTP binding proteins (G proteins). The alpha subunit of these proteins is a molecular switch, cycling between GDP-bound (inactive) and GTP-bound (active) forms. The purpose of this study is to characterize the intracellular regulation of G-protein-mediated signal transduction. GTPase activity of the alpha subunit is enhanced by a novel family of regulators of G protein signaling (RGS proteins), resulting in inhibition of Gi and Gq-coupled signaling. This project studies specifically the interaction between RGS proteins and G proteins and the resultant control of G protein function. RGS proteins demonstrate little specificity for Gi and Gq subunits in vitro, yet they aparently discriminate between G-protein- coupled receptors (GPCRs) linked to the same G-alpha in some cells. Fusion proteins consisting of different GPCRs fused to various G-alpha subunits were constructed and expressed in mammalian cell lines. Receptor-stimulated GTPase activity of membrane preparations was determined in the presence or absence of RGS proteins. RGS proteins were previously shown to augment agonist-stimulated GTPase activity of the receptor-G-alpha fusion proteins. This system is utilized to study the regulation of RGS activity by covalent modification. RGS16 was previously shown to undergo palmitoylation on conserved N-terminal cysteine residues (C2, C12). Mutation of these residues to alanine and expression in cellular membranes prevented RGS16 enhancement of serotonin-induced GTPase activity of a serotonin-G-alpha o fusion protein. Furthermore, these residues were shown to be dispensible for plasma membrane localization but reuqired for targeting to lipid rafts. N-terminal RGS16 palmitoylation was shown to permit palmitoylation of a conserved cysteine residue in the RGS box. Mutation of this residue (C98)abolished RGS16 catalytic activity in the same assay and its ability to regulation Gi-mediated adenylyl cyclase inhibition. RGS16 was shown to be phosphorylated on a conserved tyrosine residue by the receptor for Epidermal Growth Factor. This phosphorylation increased the GTPase accelerating (GAP) activity of RGS16. RGS16 was also shown to be phosphorylated on another conserved tyrosine residue by src family kinases. Such phosphorylation could be induced by stimulation of G-protein-coupled receptors or by engagement of the B cell antigen receptor in B lymphocytes transfected with RGS16. A novel interaction between the G protein G-alpha 13 and RGS16 was discovered. RGS16 overexpression inhibited G13-mediated stimulation of the small GTPase Rho, leading to impaired serum response element (SRE)-induced transcription. In addition, RGS16 injection into astrocytoma cells inhibited G13-induced Rho-dependent cell rounding. The RGS16-G13 interaction and inhibition of SRE-dependent transcription required only the RGS16 N-terminus. An interaction between endogenous RGS16 and G-alpha 13 was found in MCF7 breast carcinoma cells. Suppression of RGS16 by small interfering RNAs enhanced signaling evoked by a G-alpha 13-coupled receptor (EDG1) in MCF7 cells.